Insulin binds to its receptor on the surface of the target cell, thereby activating the insulin receptor tyrosine kinase. The activated receptor phosphorylates tyrosine residues in multiple intracellular proteins. We have investigated the downstream pathway that eventuates in translocation of GLUT4 glucose transporters to the plasma membrane in isolated rat adipocytes, a physiologically important target cell for insulin action. Using an technique involving transient expression in rat adipocytes, we have previously demonstrated that IRS-1 is one of the substrates which participates in mediating the effect of insulin to translocate GLUT4, and that activation of phosphatidyl inositol 3-kinase (PI 3-kinase) activity also plays a necessary role in mediating this action of insulin. Recently, we have extended these studies in several ways: (1) cloning the cDNA encoding murine IRS-3, another member of the insulin receptor substrate family; (2) demonstrating that murine IRS-2, murine IRS-3, and human IRS-4 can substitute for IRS-1 in mediating the translocation of GLUT4; (3) demonstrating that protein kinase B (also known as Akt) participates in this pathway downstream from PI 3-kinase. In separate studies, we have identified a family of novel proteins that associate with the insulin receptor, and also with other receptor tyrosine kinases. Sorting nexin 1 (Snx1) was originally cloned in Gill's laboratory and reported to bind to the cytoplasmic tail of the epidermal growth factor receptor. We have extended this observation by demonstrating the Snx1 also binds to several other receptors when co-expressed in mammalian cells, including the receptors for insulin, platelet-derived growth factor, leptin, and transferrin. In addition, we have cloned and characterized three additional homologs: Snx2, Snx3, and Snx4. Like Snx1, Snx2 and Snx4 associate with receptor tyrosine kinases although Snx3 does not. We are currently completing the cloning of cDNA's encoding two additional sorting nexins: Snx5 and Snx6. All six sorting nexins contain a conserved PX (NADPH oxidase homology) domain of approximately 100 amino acids. In addition, they are close homologs of several proteins (Vps5p, Grd19p, and Mvp1p) that are involved in protein trafficking in yeast. We are investigating the role of the sorting nexins in mammalian cells, with emphasis upon possible relationships to the function of insulin receptors.